Cardiovascular Tissue

Atherosclerotic vascular disease remains a leading cause of mortality and morbidity in industrialized nations. Autologous veins are the conduits of choice in the surgical creation of bypasses of short- to medium-caliber vessels in patients with peripheral occlusive arterial disease. The success rate of bypasses using conduits with diameters greater than 6 mm has been excellent, whereas the majority of bypasses using smaller conduits typically fail within 5 years. Furthermore, suitable donor sites are limited. Allogeneic grafts carry the risk of rejection and potential disease transmission, though there is recent evidence that autologous cells seeded on decellularized allogeneic vessels may provide a suitable alternative [109]. Artificial grafts face similar drawbacks in that they work reasonably well for large diameter grafts, but they have a high failure rate in small diameter grafts. Furthermore, there is a potential for infection and thrombosis. Reoperations may also be required due to calcification [110].

Due to these limitations, tissue engineering of blood vessel substitutes has become an active area of research. Early attempts focused on the creation of hybrid grafts by attempting to line the lumen of artificial graft materials with endothelial cells [111]. Subsequently, many different approaches have been taken, relying on both synthetic and natural scaffolds. A multilayered blood vessel was created in vitro by combining smooth muscle cells in collagen gel, fibroblasts, and endothelial cells. Although the histology resembled that of an artery, the mechanical properties were not sufficient for the systemic circulation [112]. A scaffold-free approach using human cultured cells resulted in increased burst strength, but the patency rate was only 50% over 7 days in an animal model [113]. A small-diameter, tissue-engineered vessel was created from small intestinal sub-mucosa and showed excellent hemostasis and patency in a rabbit arterial bypass model. Within 3 months after implantation, the grafts were remodeled into cel-lularized vessels that exhibited physiological activity in response to vasoactive agents [114]. Following the use of natural scaffolds, the use of synthetic biodegradable scaffold was assessed in a seminal study to create tissue-engineered arteries. In addition, a pulsatile flow bioreactor was used to create a more physiological environment and improve the mechanical strength of the graft. The tissue-engineered graft had the histological appearance of a native artery and was able to sustain systemic pressures. The tissue-engineered arteries were implanted in a pig model and remained patent for up to 4 weeks [115].

Repair of congenital cardiac defects frequently require large diameter conduits. As stated earlier, artificial grafts are now routinely used. However, due to the lack of growth potential, they are not suitable for pedi-atric patients. Viable pulmonary arteries were created by seeding cells derived from ovine artery and vein segments onto synthetic biodegradable PGA/PLGA scaffolds in tubular shape. These autologous constructs were used to replace a 2-cm segment of the pulmonary artery in lambs. All tissue-engineered grafts were patent and demonstrated a non-aneurysmal increase in diameter, suggesting growth [116]. A similar methodology was applied to replace a 3- to 4-cm segment of the abdominal aorta in lambs. Here, a new copolymer of PGA and polyhydroxyalkanoate (PHA) was combined with cells harvested from ovine carotid arteries. All tissue-engineered grafts remained patent, and no aneurysms had developed over a course of 3 months. Histologically, elastic fibers were observed in the medial layer, and endothelial cells lined the lumen. Furthermore, the mechanical properties of the tissue-engineered aorta approached those of the native vessel. In addition to full segment replacements, patch augmentation of vessels has also been investigated. Vascular cells isolated from ovine peripheral veins were seeded on a fast-absorbing biopolymer, poly-4-hydroxybu-tyric acid (P4HB), and assessed for patch augmentation of the proximal pulmonary artery in a juvenile sheep model. Postoperative echocardiography showed no signs of dilatation or stenosis. Macroscopically, a smooth internal surface with increasing tissue formation was observed [117]. Another study demonstrated the successful replacement of the inferior vena cava in a dog model. In this approach, mixed cells obtained from the femoral veins of mongrel dogs were seeded onto tube-shaped biodegradable polymer scaffolds composed of a PGA nonwoven sheet and a polycap-rolactone-polylactide copolymer (PCLA). No implants showed evidence of dilatation or stenosis. In addition, an endothelial lining was observed in all tissue-engineered grafts [118].

An important milestone for tissue engineering was achieved in May 2000, when an occluded pulmonary artery was successfully replaced by a tissue-engineered graft in a 4-year-old girl with a single ventricle and pulmonary atresia that had previously undergone pulmonary artery angioplasty and the Fontan procedure. Following harvest of a short segment of peripheral vein, cells were isolated, cultured in vitro and seeded on a tubular PCLA scaffold. Ten days after seeding, the graft was transplanted. Seven months after implantation, the patient was doing well. Chest radiography revealed no evidence of graft occlusion or aneurysmal changes [119]. The same methodology was applied in a subsequent case to replace an occluded Dacron graft in the extracardiac Fontan operation (ECFO) of a 12-year-old boy. The reoperative ECFO with a tissue-engineered graft was successful, and postoperative computed tomography done 4 months after the operation revealed a patent graft [120]. Subsequently, aspirated bone marrow cells were used as the cell source and seeded on the scaffold on the day of surgery. Using this method, sufficient cells could be obtained on the day of the surgery without requiring a culturing period. Furthermore, extra hospitalization for vein harvesting is not required. This approach has been applied in 22 patients, and good results were obtained after surgery [121]. The contribution of bone marrow cells to the histogenesis of autologous tissue-engineered vascular grafts was also demonstrated [122]. In addition, a new technique of extracardiac total cavopulmonary connection using a tissue-engineered graft has been developed, and the initial results are promising (Fig. 16.5) [123].

Tissue-engineered vascular grafts have shown promise in the aforementioned complicated cases. It is anticipated that their indications will be increased for other types of cases. While the initial clinical success is very promising, there remain limitations of this method in a clinical setting. First, these tissue-engineered grafts are currently limited to the pulmonary circulation. Tissue-engineered grafts cannot be used in the systemic

Fig. 16.5 Angiograms of tissue-engineered grafts 6 months after undergoing an extracardiac total cavopulmonary connection (TCPC) operation in Patient 2 (a), 4 months after TCPC in Patient 3 (b), 1 month after TCPC in Patient 4 (c), and 8

months after TCPC in Patient 6 (d). Note the smooth surface of the tissue-engineered graft and well-enhanced pulmonary arteries. (Reprinted with permission from [123])

Fig. 16.5 Angiograms of tissue-engineered grafts 6 months after undergoing an extracardiac total cavopulmonary connection (TCPC) operation in Patient 2 (a), 4 months after TCPC in Patient 3 (b), 1 month after TCPC in Patient 4 (c), and 8

months after TCPC in Patient 6 (d). Note the smooth surface of the tissue-engineered graft and well-enhanced pulmonary arteries. (Reprinted with permission from [123])

circulation due to the higher pressures and flow velocities. To achieve this goal, further development of biodegradable scaffolds is required. Second, a tissue-engineered graft cannot be used in an emergency operation due to the prolonged in vitro period necessary for cell engraftment on the scaffold. Third, a sufficient cell number may not be available in all patients. In such cases, additional cell sources from other parts of the body must be assessed. Active research continues in this field to overcome these challenges. Efforts are devoted to modifying the culture environment to enhance extracellular matrix synthesis and organization using bioreactors under physiologic conditions and biochemical supplements. Improved understanding of the factors involved in cardiovascular development and advances in gene therapy and stem cell biology are also expected to contribute toward the goal of widespread clinical applications.

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